Cytotoxic T lymphocytes (CTL) contribute to the clearance of many viruses such as CMV, EBV, HBV and influenza virus. Although CTL have also been isolated from the peripheral blood and liver of HCV-infected patients, their role in the resolution of disease is still unclear. During the last year, we have continued to build on our previous observation, at which the HCV-specific T cell response is more vigorous after recovery than in chronic HCV infection. Specifically, we have determined the vigor and specificity of the cellular response with chromium release assays after in vitro stimulation of PBMC with HLA-A2 restricted peptides and the quantity of HCV specific cells by staining unstimulated PBMC with peptide-MHC tetramers and by limiting dilution cultures. In total, 8 recovered patients, 10 chronic patients with and 10 chronic patients without elevated liver enzymes, and 8 healthy controls were studied, all of whom were HLA-A2 positive. Our results demonstrate that the difference in the strength of the CTL response between recovered and chronic patients seems to be due to a vigorous and multispecific recall response upon exposure to the specific antigen rather than to the frequency of circulating HCV-specific CD8+ T cells. While the recall response upon reexposure to antigen was more vigorous in recovered than in chronic patients, the number of antigen-specific cells prior to in vitro expansion was lower in recovered than in chronic patients. The results also suggest that a subgroup of epitope specific T cells in the blood of chronically infected patients does not display cytotoxic effector functions and / or do not expand upon in vitro stimulation. This aspect will be analyzed further.In a second, related study, we have crossed HLA-A2 transgenic mice with HCV transgenic mice in order to characterize the intrahepatic effects of the HLA-A2 restricted CTL response. Mice were immunized with an HLA-A2 restricted peptide from the HCV core protein, frequently recognized by HLA-A2 positive patients in the study described above. This peptide represents an immunodominant epitope, since immunization with peptide as well as with vaccinia virus encoding the HCV core protein induced epitope specific CTL in HLA-A2 transgenic mice. When HLA-A2 x HCV transgenic mice were immunized with the peptide, immunological tolerance against the transgene could be broken in 7/9 mice that displayed strong CTL responses (mean specific cytotoxicity: 32%, range 13-59%). Interestingly, only 1/7 mice displayed histologic evidence of intrahepatic periportal inflammation and none of the mice developed elevated ALT levels. - HCV/CTL/Immune Response/Immunization/Tolerance - Human Subjects